Abstract
Objective Neutral endopeptidase (NEP, EC 3.4.24.11) metabolises endogenous vasoactive peptides that may protect against atherogenesis. Since NEP is found in the adventitia of arteries, we investigated the anti-atherogenic effects of chronic adventitial NEP inhibition.
Methods Intimal hyperplasia of rabbit carotid arteries was induced by placement of soft, non-occlusive, peri-arterial silastic collars. NEP localisation was studied with autoradiography 7 and 14 days after collar placement. Vascular NEP was inhibited in vivo by local superfusion of one collared carotid artery with Candoxatrilat (50 pmol/h), for 7 days (n=7). The contralateral collar was filled with saline vehicle. After 7 days, ring segments of collared and normal (proximal to the collar) arteries were obtained and in vitro functional measurements, immunohistochemical determination of the pro-atherogenic factor plasminogen activator inhibitor-1 (PAI-1), localization of macrophages and morphometric analyses were carried out.
Results Vascular NEP radiolabelled substrate binding, mainly in the media, was increased by ∼50% after 7 days (n=5; p<0.05) and by ∼300% after 14 days of collar placement (n=5; p<0.05). Compared with normal artery segments from the same animal, vehicle-filled collared sections displayed significantly impaired vasorelaxation to acetylcholine (endothelium-independent vasodilatation was preserved), increased PAI-1 immunostaining, macrophage accumulation and intimal thickening. In Candoxatrilat-treated collared arteries, vasorelaxation to acetylcholine was improved, along with reductions in PAI-1 levels, macrophage numbers and intimal area (all p<0.05).
Conclusion Enhancing the activity of local, endogenous peptides by adventitial inhibition of vascular NEP may protect against early atherogenesis. This is of particular relevance to using adventitial therapies to prevent intimal hyperplasia leading to restenosis.
1. Introduction
The complex vascular reaction to injury, known as atherogenesis, involves blood-borne and local vascular components [1]. Intimal hyperplasia, inflammation and endothelial dysfunction are important features in the vascular response to injury. Previously, pharmacological interventions to quell atherogenesis have been applied from the luminal surface. This has been either via the circulation or, more recently, from coated stents. An alternative approach, however, is to focus on pro-atherogenic mechanisms that lie within the arterial wall. Indeed, the adventitial region may be an important source of the cells and signals causing neointima formation and arterial lumen narrowing that underlie the important clinical condition of restenosis. Several studies, in pigs [2,3], rats [4] and mice [5], have demonstrated that adventitial fibroblasts can migrate to the developing neointima where they may undergo phenotypic conversion to myofibroblasts and contribute to intimal thickening. As such, the adventitia has been proposed as a target for delivery of anti-atherogenic, anti-restenotic treatment.
Our interests are in the endogenous protective factors, peptides in particular, that derive from within the blood vessel wall itself and which may counteract pro-inflammatory and pro-atherogenic forces. We have recently shown that when C-type natriuretic peptide (CNP), the natriuretic peptide made in the endothelium, is administered from the adventitial surface of carotid arteries, in vivo, important components of the atherogenic response to carotid arterial collar placement in rabbits are reduced [6,7]. In this model of peri-vascular inflammation induced by the presence of the non-occlusive, silastic collar [8,9], the endothelium remains intact but dysfunctional. There is significant intimal hyperplasia, plasminogen activator inhibitor-1 (PAI-1), a factor known to be involved in extracellular matrix production, is activated and macrophages accumulate, particularly in the adventitia [7]. Although infused CNP reduces PAI-1 and macrophage accumulation and improves endothelial function in collared arteries, effects of the peptide on intimal thickening are small. We have proposed [7] that the beneficial effects of CNP may be limited by the upregulation of neutral endopeptidase 24.11 (NEP), the degradative enzyme contained within the vascular wall.
NEP is a membrane bound enzyme that metabolises the natriuretic peptides, among other substrates, and appears in vascular smooth muscle cells (VSMC), vascular endothelial cells [10,11] and has been identified in all layers of the normal vascular wall [12]. Although intravenous inhibition of NEP has been shown to be protective in a rabbit model of lipid-loaded atherosclerosis [13], the role of NEP from within the arterial wall and the effect of NEP inhibition from the adventitial surface have not been investigated. Moreover, due to the lack of substrate specificity of NEP [14], the systemic inhibition of NEP may result in enhanced activity of other peptides that promote undesirable effects such as neurogenic inflammation mediated by calcitonin gene-related peptide (CGRP) [15]. Thus, a more targeted, tissue-specific approach may be warranted. The present study was designed to determine whether manipulation of endogenous, adventitial NEP influences the development of intimal hyperplasia. In the current study, the localisation of and changes in NEP were investigated in rabbit arteries collared for 7 or 14 days to induce intimal thickening. The effect of adventitial NEP inhibition on vascular reactivity, morphology, PAI-1 and macrophage accumulation was also examined in collared arteries after 7 days.
2 Methods
2.1 General procedures
Male New Zealand white rabbits, weighing between 3.0 and 4.0 kg, were maintained on normal laboratory chow (zero cholesterol). Rabbits were anaesthetised with Propofol (i.v. 0.5 mg/kg; Abbott Australasia) followed by intramuscular injection containing Xylazine (20 mg/kg; Xylaze 100, Parnell Laboratories, Australia) and Ketamine (100 mg/kg; Ketapex, Apex Laboratories, Australia). A soft, non-occlusive, silastic collar was placed around each common carotid artery as previously described (e.g., [6–8]). All procedures were approved by the Howard Florey Institute Animal Ethics Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
Experiments were conducted in 3 groups of rabbits. Collars were filled with 0.9% saline and arteries were collected 7 days (Group I, n=5) or 14 days (Group II, n=5) after collar placement. An additional group was treated with the NEP inhibitor, Candoxatrilat (50 μmol/l in 0.9% saline; provided by Pfizer Central Research, Sandwich, UK), infused directly into one collar via an osmotic mini-pump (1 μl/h, Model 2001; Alza Pharmaceuticals, USA; method previously described [7]) from the time of collar placement for 7 days (Group III, n=7). The contralateral collar was filled with 0.9% saline. Candoxatrilat was originally shown to be specific for NEP [16] and does not cross-react with ECE-1 [17].
At the end of the study, rabbits were heparinised (1500 IU, i.v.) and killed with an overdose of sodium pentobarbitone (100 mg/kg i.v.; Nembutal, Merial Australia Pty Ltd). Both carotid arteries were removed and the region of artery within each collar, along with an equal sized segment of ‘normal’ artery proximal to the collar from the same vessel, were dissected in Kreb's bicarbonate solution (pH 7.4) as previously described [6,9].
2.2 Autoradiographic localization of NEP (Groups I and II)
In vitro autoradiography to localise NEP was performed on slide-mounted, 14 μm sections of one ring (∼3 mm in length) from the centre of each arterial segment, as previously described [18]. The radiolabelled inhibitor of NEP used was 125I-SCH 47896 (ProSearch International Australia P/L, Australia [19]) and is referred to as the NEP radiolabelled substrate. Non-specific binding (NSB) was determined in the presence of 100 mmol/l Na2EDTA and 2.5 mmol/l phenanthroline.
2.3 Vascular reactivity of Candoxatrilat-treated arteries (Group III)
Arterial rings from normal and collared segments of each vessel were suspended in organ baths for isometric tension recording [6]. A cumulative concentration-response curve to 5-hydroxytryptamine (5-HT, 0.01 to 10 μmol/l; Sigma, USA) was constructed to determine maximum contraction for each ring. The vessels were then washed and left for 45 min. Arterial rings were pre-contracted submaximally with 5-HT to achieve 70% of maximum contraction, and a full concentration–response curve to acetylcholine (ACh, range 0.001 to 10 μmol/l; BDH Chemicals, UK), as the endothelium-dependent vasodilator, was obtained. All tissues were then washed and left for 30 min. They were again pre-contracted submaximally with 5-HT and a full concentration–response curve to CNP (range 0.0001 to 1 μmol/l) was obtained. It has been demonstrated previously that endothelium-independent relaxation to sodium nitroprusside is not reduced in rabbit carotid arteries collared for 7 days [6]. In the present study, CNP was used as another endothelium-independent vasorelaxant and was used to determine whether chronic NEP inhibition in vivo influenced CNP's vasorelaxant actions on the collared arteries in vitro.
Maxima and pEC50 values were compared from the 5-HT and ACh concentration–response curves. Since the CNP concentration–response did not reach maximum relaxation, maxima could not be compared. Instead, the slopes of the responses to CNP were calculated for each rabbit using linear regression over the dose range 10− 9 to 10− 6 M and grouped data were compared.
2.4 Histology and morphometric analyses
Intimal thickening in all collared arteries and normal histology of uncollared sections were confirmed by light microscopy of haematoxylin and eosin-stained sections (details described in Barber et al. [6]). Morphometric analyses of intimal and medial areas were carried out on all arterial sections from rabbits treated with Candoxatrilat (Group III), as previously described [6].
2.5 Localisation and quantification of PAI-1 and localisation of macrophages (Group III)
The effect of NEP inhibition on vascular PAI-1 immunoreactivity was investigated in rabbits from Group III using sheep anti-PAI-1 antiserum, as previously described [7]. Comparative levels of PAI-1 were determined by densitometry of the 4 samples from each rabbit that were processed and measured under identical conditions, as previously described [7]. Results are reported as relative optical density (ROD) and normalised to the uncollared (normal) segment of saline-collared artery.
Macrophages were localised by mouse anti-rabbit alveolar macrophage (RAM-11) immunohistochemistry, as previously described [7]. For macrophage immunohistochemistry, serial sections of 4 μm were cut immediately adjacent to the sections used for PAI-1 measurements. Macrophages in three sections from each tissue, each section separated by at least 5 sections, were counted by an observer blinded to the treatment group. To normalise the variance, the macrophage data were log transformed for statistical analysis.
2.6 Data analysis and statistics
All data were expressed as means±S.E.M., and the reported n values refer to the number of rabbits. In all cases, probabilities of p<0.05 were considered to be statistically significant. Unless otherwise stated, SigmaStat (SPSS Science, Chicago, IL) was used for all statistical comparisons. Paired t-tests were used to compare NEP radiolabelled substrate binding in normal and collared carotid artery sections, to compare intimal areas and intimal PAI-1 levels between saline-collared arteries and Candoxatrilat-treated collared arteries, and to compare the effect of Candoxatrilat on numbers of macrophages (after conversion to log10 to normalise the variance). In all other cases, statistical analyses were made using a 2-way ANOVA with repeated measures and pair-wise multiple comparisons using Tukey's test.
3 Results
3.1 Autoradiography (Groups I and II)
NEP radiolabelled substrate binding was detected in all carotid artery sections. No significant non-specific binding was detected.
3.2 7-day peri-arterial collars (Group I)
By comparison with haematoxylin and eosin-stained sections from the same segment, NEP radiolabelled substrate binding was primarily localised to the media, where it appeared evenly distributed, and adventitia of normal artery sections. In collared arteries, binding across the media, intima and endothelium of the artery appeared to be stronger than adventitial binding. NEP radiolabelled substrate binding was increased (p<0.05) in collared artery sections when compared to sections of normal artery (Fig. 1A).
NEP binding values of 125I-SCH47896 in rabbit carotid arteries. Total binding data are expressed in disintegrations per minute per mm2 of tissue (means±S.E.M.) for each artery ring. Normal (open bars) and collared (black bars) carotid arteries were collected from rabbits 7 days (A, n=5) or 14 days (B, n=5) after surgery to implant peri-arterial collars. *p<0.05 significant effect of collar placement.
NEP binding values of 125I-SCH47896 in rabbit carotid arteries. Total binding data are expressed in disintegrations per minute per mm2 of tissue (means±S.E.M.) for each artery ring. Normal (open bars) and collared (black bars) carotid arteries were collected from rabbits 7 days (A, n=5) or 14 days (B, n=5) after surgery to implant peri-arterial collars. *p<0.05 significant effect of collar placement.
3.3 14-day peri-arterial collars (Group II)
Normal artery sections from rabbits with 14-day saline-filled collars displayed the same NEP radiolabelled substrate binding pattern as normal sections from rabbits with 7-day saline-filled collars (Fig. 2A and C). Arteries collared for 14 days displayed a strong punctate binding pattern in the adventitia and extra-adventitial layers in addition to even binding throughout the media (Fig. 2B and D). NEP radiolabelled substrate binding in 14-day collared arteries was 3 times the binding in normal artery sections from the same vessels (p<0.05, Fig. 1B). The increase in NEP radiolabelled substrate binding in 14-day collared arteries was significantly greater than the increase seen in collared arteries after just 7 days (% increase in collared artery compared to normal artery: 63±9% after 14 days vs. 18±6% after 7 days; p<0.01). The presence of Na2EDTA and phenanthroline completely prevented binding of 125I-SCH47896 (Fig. 2E and F; NSB).
NEP binding of 125I-SCH47896 in carotid arteries from one rabbit, 14 days after implantation of peri-arterial collars. Slides were opposed to film and then sections stained with haematoxylin and eosin: normal (A) and collared artery (B) sections. Digitised autoradiographs illustrate total binding in normal (C) and collared (D) artery sections. Non-specific binding in normal (E) and collared (F) artery sections is defined in Methods. Scale bars (A and B) represent 200 μm. Arrows (A and B) indicate the internal elastic lamina.
NEP binding of 125I-SCH47896 in carotid arteries from one rabbit, 14 days after implantation of peri-arterial collars. Slides were opposed to film and then sections stained with haematoxylin and eosin: normal (A) and collared artery (B) sections. Digitised autoradiographs illustrate total binding in normal (C) and collared (D) artery sections. Non-specific binding in normal (E) and collared (F) artery sections is defined in Methods. Scale bars (A and B) represent 200 μm. Arrows (A and B) indicate the internal elastic lamina.
3.4 Vascular reactivity (Group III)
Cumulative addition of 5-HT caused concentration-dependent vasocontraction of all isolated carotid artery rings. As previously described [9], collared artery rings were more sensitive to 5-HT than normal artery rings; illustrated by the higher pEC50 values (Table 1) of collared arteries (p<0.05, both saline- and Candoxatrilat-treated). The difference in maximum vasocontraction, however, did not quite reach significance (p=0.07). Treatment of collared arteries with peri-arterial Candoxatrilat (50 pmol/h) did not alter the sensitivity of collared arteries to 5-HT (Table 1).
5-HT responses in isolated rabbit carotid artery rings
| Artery | Maxima | pEC50 |
|---|---|---|
| Normal (for saline-collared artery) | 2.8±0.4 | 7.11±0.07 |
| Saline-collared artery | 3.9±0.6 | 7.47±0.09* |
| Normal (for Candoxatrilat-collared artery) | 2.6±0.3 | 7.16±0.04 |
| Candoxatrilat-collared artery | 4.2±0.5 | 7.50±0.08* |
| Artery | Maxima | pEC50 |
|---|---|---|
| Normal (for saline-collared artery) | 2.8±0.4 | 7.11±0.07 |
| Saline-collared artery | 3.9±0.6 | 7.47±0.09* |
| Normal (for Candoxatrilat-collared artery) | 2.6±0.3 | 7.16±0.04 |
| Candoxatrilat-collared artery | 4.2±0.5 | 7.50±0.08* |
A collar was placed around each carotid artery for 7 days (n=7). One collar was treated with the NEP inhibitor Candoxatrilat (50 pmol/h) and the contralateral collar was filled with saline vehicle. Normal carotid artery segments were collected from each vessel, outside and proximal to the collar.
p<0.05 vs. normal artery from the same vessel, 2-way RM ANOVA.
5-HT responses in isolated rabbit carotid artery rings
| Artery | Maxima | pEC50 |
|---|---|---|
| Normal (for saline-collared artery) | 2.8±0.4 | 7.11±0.07 |
| Saline-collared artery | 3.9±0.6 | 7.47±0.09* |
| Normal (for Candoxatrilat-collared artery) | 2.6±0.3 | 7.16±0.04 |
| Candoxatrilat-collared artery | 4.2±0.5 | 7.50±0.08* |
| Artery | Maxima | pEC50 |
|---|---|---|
| Normal (for saline-collared artery) | 2.8±0.4 | 7.11±0.07 |
| Saline-collared artery | 3.9±0.6 | 7.47±0.09* |
| Normal (for Candoxatrilat-collared artery) | 2.6±0.3 | 7.16±0.04 |
| Candoxatrilat-collared artery | 4.2±0.5 | 7.50±0.08* |
A collar was placed around each carotid artery for 7 days (n=7). One collar was treated with the NEP inhibitor Candoxatrilat (50 pmol/h) and the contralateral collar was filled with saline vehicle. Normal carotid artery segments were collected from each vessel, outside and proximal to the collar.
p<0.05 vs. normal artery from the same vessel, 2-way RM ANOVA.
ACh caused concentration-dependent relaxation of all carotid artery rings (Fig. 3A). The endothelium-dependent relaxation responses to ACh in saline-collared artery rings were impaired compared to normal artery rings from the same vessel (Fig. 3A, closed circles vs. open circles). This impairment was evident as a rightward shift (p<0.05) of the concentration–response curves (Fig. 3A, pEC50). Local Candoxatrilat treatment for 7 days improved endothelium-dependent relaxation of collared arteries. The maximum relaxation response to ACh of Candoxatrilat-treated collared arteries was not different from the response of normal artery rings from the same vessel (Fig. 3A, closed squares vs. open squares). The concentration–response curve of Candoxatrilat-treated collared arteries was shifted to the left (p<0.05) of saline-collared arteries (Fig. 3A, pEC50, closed squares vs. closed circles). The pEC50 values of Candoxatrilat-treated rings remained lower (p<0.05) than the normal rings from the same vessel (Fig. 3A).
Vasorelaxation to ACh (A) or CNP (B) in rabbit carotid arteries, expressed as % relaxation (means±S.E.M.) in each tissue following submaximal (70%) pre-contraction with 5-HT. Normal and collared carotid artery segments were collected from rabbits (n=7) 7 days after implantation of peri-arterial collars. One collar was superfused with Candoxatrilat (50 pmol/h). The contralateral collar was filled with saline. Functional measurements were carried out on four different tissues from each rabbit: saline-collared artery (•), normal artery proximal to the collar (О), Candoxatrilat-treated collared artery (■), and normal artery proximal to the collar (□). Symbols (all p<0.05) in panel A are as follows: *maximum relaxation of saline-collared arteries vs. all others; #pEC50 values from saline-collared artery vs. normal artery rings from the same vessel; †pEC50 values from Candoxatrilat-treated collared artery vs. normal artery rings from the same vessel; ‡pEC50 values from Candoxatrilat-treated collared artery vs. saline-collared artery rings. Symbol (p<0.05) in panel B: *slope of the curve from collared artery vs. normal artery rings from the same vessel.
Vasorelaxation to ACh (A) or CNP (B) in rabbit carotid arteries, expressed as % relaxation (means±S.E.M.) in each tissue following submaximal (70%) pre-contraction with 5-HT. Normal and collared carotid artery segments were collected from rabbits (n=7) 7 days after implantation of peri-arterial collars. One collar was superfused with Candoxatrilat (50 pmol/h). The contralateral collar was filled with saline. Functional measurements were carried out on four different tissues from each rabbit: saline-collared artery (•), normal artery proximal to the collar (О), Candoxatrilat-treated collared artery (■), and normal artery proximal to the collar (□). Symbols (all p<0.05) in panel A are as follows: *maximum relaxation of saline-collared arteries vs. all others; #pEC50 values from saline-collared artery vs. normal artery rings from the same vessel; †pEC50 values from Candoxatrilat-treated collared artery vs. normal artery rings from the same vessel; ‡pEC50 values from Candoxatrilat-treated collared artery vs. saline-collared artery rings. Symbol (p<0.05) in panel B: *slope of the curve from collared artery vs. normal artery rings from the same vessel.
Cumulative addition of CNP caused concentration-dependent relaxation of all carotid artery rings (Fig. 3B). Unlike the endothelium-dependent vasorelaxation to ACh in collared vessels (Fig. 3A), the concentration–response vasorelaxation to endothelium-independent CNP was not reduced in collared vessels (Fig. 3B). Indeed, the slope was steeper (p<0.05) in collared artery rings compared to normal artery rings (Fig. 3B). Candoxatrilat did not affect relaxation to CNP in normal or collared carotid artery rings (Fig. 3B).
3.5 Morphometry (Group III)
Normal artery sections from proximal to the collar displayed no disturbance in vascular structure (Fig. 4A and B). Placement of collars around the carotid arteries caused substantial intimal thickening (Fig. 4C and D). Collared arteries treated locally with Candoxatrilat displayed >50% reduction in intimal thickening (Fig. 4D vs. C). Intimal area was reduced (p<0.05) from 79.2±20.2 in saline-collared arteries to 34.9±7.4 (× 1000 μm2) in Candoxatrilat-treated collared arteries, with accompanying reductions in the intima/media ratio (Fig. 5A).
Panel A depicts the intima/media ratio (IMR, cross-sectional area of intima divided by cross-sectional area of media) of collared carotid arteries. Panels B–E depict the relative optical density (ROD) of PAI-1 immunostaining in carotid arteries. Tissues from each rabbit (n=7) were: saline-collared artery (striped bars), normal artery proximal to the saline-collar (open bars), Candoxatrilat-treated collared artery (hatched bars) and normal artery proximal to the Candoxatrilat-treated collar (black bars). PAI-1 levels (means±S.E.M.) were measured in each layer of the vascular wall separately: (B) endothelium, (C) neointima, (D) media and (E) adventitia. The percentages (panels B, D and E) are the within animal mean PAI-1 ROD for each tissue normalised to the uncollared (saline side) section of normal artery (100%). For the intimal region (panel C), saline-collared artery was deemed 100%. Since the sub-endothelial, intimal layer of normal (uncollared) rabbit carotid arteries was too small to accurately measure at 20 × magnification, there are no IMR or intimal PAI-1 values for normal carotid arteries. †p<0.05 vs. saline-collared arteries; *p<0.05 vs. normal (saline) arteries.
Panel A depicts the intima/media ratio (IMR, cross-sectional area of intima divided by cross-sectional area of media) of collared carotid arteries. Panels B–E depict the relative optical density (ROD) of PAI-1 immunostaining in carotid arteries. Tissues from each rabbit (n=7) were: saline-collared artery (striped bars), normal artery proximal to the saline-collar (open bars), Candoxatrilat-treated collared artery (hatched bars) and normal artery proximal to the Candoxatrilat-treated collar (black bars). PAI-1 levels (means±S.E.M.) were measured in each layer of the vascular wall separately: (B) endothelium, (C) neointima, (D) media and (E) adventitia. The percentages (panels B, D and E) are the within animal mean PAI-1 ROD for each tissue normalised to the uncollared (saline side) section of normal artery (100%). For the intimal region (panel C), saline-collared artery was deemed 100%. Since the sub-endothelial, intimal layer of normal (uncollared) rabbit carotid arteries was too small to accurately measure at 20 × magnification, there are no IMR or intimal PAI-1 values for normal carotid arteries. †p<0.05 vs. saline-collared arteries; *p<0.05 vs. normal (saline) arteries.
Light microscopy images of transverse sections of carotid arteries from a single rabbit, collected 7 days after implantation of peri-arterial collars. Haematoxylin and eosin stained sections are normal artery from outside the collar (A and B); and artery from within the saline-filled collar (C) or within the Candoxatrilat-treated collar (D). Panels E and F illustrate photomicrographs of PAI-1 immunostaining in the saline-collared (E) and the Candoxatrilat-treated collared (F) artery from the same rabbit. Strong PAI-1 immunoreactivity is present in the endothelium and the intima of the collared arteries. The white arrows indicate the internal elastic lamina; the black arrows indicate the external elastic lamina.
Light microscopy images of transverse sections of carotid arteries from a single rabbit, collected 7 days after implantation of peri-arterial collars. Haematoxylin and eosin stained sections are normal artery from outside the collar (A and B); and artery from within the saline-filled collar (C) or within the Candoxatrilat-treated collar (D). Panels E and F illustrate photomicrographs of PAI-1 immunostaining in the saline-collared (E) and the Candoxatrilat-treated collared (F) artery from the same rabbit. Strong PAI-1 immunoreactivity is present in the endothelium and the intima of the collared arteries. The white arrows indicate the internal elastic lamina; the black arrows indicate the external elastic lamina.
3.6 PAI-1 immunohistochemistry (Group III)
PAI-1 immunoreactivity was detected in all layers of the arterial wall, with the most intense staining in the endothelium (Fig. 4E and F and Fig. 5B–E). Compared to normal segments from the same artery, PAI-1 staining was increased in the endothelium (Fig. 5B; p<0.05) and adventitia (Fig. 5E; p<0.05) of saline-collared arteries; however, the increase in the media (Fig. 5D) did not reach significance. PAI-1 was also observed in the intima of saline-collared artery sections (Fig. 4E). In collared arteries, local Candoxatrilat treatment significantly reduced PAI-1 levels in all layers of the artery wall, including the intima, compared to saline-collared arteries (Fig. 5B–E).
3.7 Macrophages (Group III)
RAM-11 immunoreactive macrophages were only observed in collared arteries. In saline-collared segments (for 7 days), macrophages accumulated in the adventitia but rarely infiltrated into the media and were not observed in the intimal region. Candoxatrilat treatment reduced the appearance of macrophages in 6 of the 7 rabbits with group data showing a 55% reduction (numbers of macrophages per arterial segment were 5.6±2.1, saline collar vs. 2.5±1.2, Candoxatrilat collar; p<0.05).
4 Discussion
The findings of this study demonstrate that local NEP within the arterial wall promotes the atherogenic process. Inhibition of NEP, by the application of a NEP inhibitor to the adventitial surface of collared carotid arteries, improved endothelial function, prevented intimal thickening and reduced PAI-1 immunoreactivity and macrophage accumulation. NEP substrate binding within the arterial wall increases along with the development of intimal hyperplasia and this upregulation of NEP is progressive, increasing with the time that the collar remains in situ. The increased levels of NEP are likely to diminish the levels of endogenous CNP and hence accelerate the rate of neointimal formation. NEP may therefore be an important therapeutic target in the search for strategies to treat or prevent intimal hyperplasia leading to restenosis.
NEP protein and enzyme activity is highest in the adventitia of rat aorta, followed by the media and lowest in the endothelium [20]. In normal rabbit carotid arteries and in 7-day collared arteries, NEP was mainly localised to the media. After 14 days, however, collared arteries displayed a punctate binding pattern of radiolabelled substrate in the adventitia, which had a very high NEP binding density. This increase in binding may be associated with an accumulation of cytokines in the collar, thought to contribute to the effects of collaring [21]. The cytokine, transforming growth factor (TGF)-β, increases NEP in renal VSMC [22].
In the current study, local NEP inhibition for just 1 week reduced the size of the intimal area by 50%. Intimal thickening is one of the important processes that underlies restenosis [23], the re-narrowing of the artery following angioplasty or the application of a stent to an atherosclerotic artery. Although the introduction of drug-eluting stents has reduced restenosis rates, there remain significant problems of neointima formation associated with in-stent restenosis [24].
It was surprising that the in vitro vasorelaxation response to CNP was not reduced in collared vessels that had been chronically treated with the NEP inhibitor. One may have predicted that the presence of the NEP inhibitor in vivo would prevent the breakdown of endogenous CNP, resulting in elevated tissue CNP levels. In turn, this may downregulate CNP receptors on VSMC (NPB guanylate cyclase receptors [25]), resulting in an attenuated vasodilator response to CNP in vitro. It is possible however that a small amount of Candoxatrilat may have survived the organ bath washing, continuing to inhibit the breakdown of peptides in vitro. Recently it was shown that the addition of a NEP inhibitor to porcine coronary arteries in vitro resulted in significantly greater relaxation response to added CNP [26], an effect independent of the endothelium. Thus, residual NEP inhibition could have counteracted any change in NPB receptor activity.
The functional, immunohistochemical and morphological effects of adventitial NEP inhibition in the present study closely parallel those of adventitial CNP administration to collared rabbit carotid arteries [6,7] or when the CNP gene was transfected into balloon-injured rabbit carotid arteries [27]. If CNP is the primary substrate for NEP then the beneficial effects of NEP inhibition on proliferation and inflammatory processes may be via increased levels of CNP acting through NPB or NPC receptors. Activation of these receptors has been shown in vitro to inhibit proliferation of VSMC [28]. Moreover, in rats and rabbits in vivo, using compression injury that damages the endothelium and promotes intimal hyperplasia, the NPC receptor appears to be selectively upregulated in the newly formed neointima [29,30]. It is therefore possible that upregulated NPC receptors, that are also the so-called “clearance receptors”, contribute to the removal of endogenous CNP along with NEP. Whether the cellular target for CNP's actions is directly on VSMC, or indirectly via fibroblasts in the adventitia, is yet to be determined.
Although it is tempting to speculate that the anti-atherogenic effects of NEP inhibition are via CNP, NEP inactivates several other vasoactive peptides that are also made in the vascular wall including endothelin, substance P, kinins and adrenomedullin [14]. Like CNP, adrenomedullin inhibits VSMC migration [31] and proliferation [32] in vitro and also inhibits ANG II-induced upregulation of PAI-1 mRNA and protein in cultured rat endothelial cells [33]. Thus, CNP or adrenomedullin, or both, may be responsible for the beneficial effects of NEP inhibition observed in the current study. Of the other NEP substrates that are located within the arterial wall, endothelin (ET) is an unlikely candidate because it has atherogenic rather than anti-atherogenic properties [34]. Indeed, systemic NEP inhibition in hypercholesterolaemic rabbits reduced tissue ECE-1, the enzyme that converts big ET-1 to the active peptide ET-1, although circulating or tissue ET-1 levels did not change [17]. Substance P has been localised to vascular endothelial cells [35], but this peptide has not been reported to have anti-atherogenic properties. Kinins such as bradykinin stimulate endothelial release of NO and prostacyclin which subsequently inhibit VSMC proliferation [36] and may contribute to anti-atherogenic effects of local NEP inhibition. Regardless of the substrate, our experiments demonstrate that inhibition of adventitial NEP is a novel target for vascular therapy.
The present study supports previous experimental findings of a beneficial effect of systemic NEP inhibition in atherosclerotic rabbits on vascular function and aortic atheroma area [13,17,37]. Those findings are now extended to the normocholesterolaemic condition and include not only effects on endothelial dysfunction, but also on intimal area, PAI-1 accumulation and macrophage infiltration. The tissue-selective inhibition of adventitial NEP may be an important advantage over the less-specific systemic NEP inhibition of earlier studies. Since NEP acts on a range of peptides that have actions not confined to vascular growth and function, systemic inhibition of NEP could result in unwanted actions as diverse as promotion of neurogenic inflammation through CGRP [15], facilitation of tumour growth via VIP (as shown in cell culture of human neuroblastoma cells [38]) and vasoconstriction through endothelin (as demonstrated in the human forearm [39]).
The atherogenic response to peri-arterial collar placement results from a stimulus to the outside of the vessel rather than a disruption or injury to the luminal surface, as occurs with restenosis after angioplasty and stenting. Nevertheless, there are strong similarities of the collared artery to those of the stented vessel undergoing restenosis-namely neointima formation and inflammation. The collar model allows for adventitial administration of drugs without the influence from potential side effects or haemodynamic changes arising from systemic treatments. An important difference is in the endothelial layer which is substantially damaged in the clinical condition but remains intact in collared vessels. This is also an advantage of the model, however, because it allows direct measurements of endothelial function that are not possible in de-endothelialised vessels often used to study atherogenesis. An intriguing question that arises from the present study is whether inhibition of adventitial NEP enhances re-endothelialisation, as may be predicted if NEP inhibition works through CNP. Along with its anti-proliferative actions on VSMC, CNP promotes endothelial regeneration following injury [40]. Whether these findings in the rabbit will apply in the human condition is unknown at present, but since NEP-like enzymes are ubiquitous across species [41], and the anti-atherogenic effects of CNP have been demonstrated in rat (e.g., [42]), rabbit [6,7] and pig [43], it is likely that the same beneficial effects of the peptide would appear in humans.
In summary, NEP activity and expression are increased in an animal model of intimal hyperplasia. Adventitial NEP inhibition reduces the progression of collar-induced atherogenesis. The effectiveness of NEP inhibition when administered locally indicates that vascular NEP contributed to the process of atherogenesis. The beneficial effects of local NEP inhibition, improving endothelial function, decreasing PAI-1 and reducing intimal thickening and macrophage accumulation, provide strong support for NEP inhibition as an effective treatment for restenosis in humans. Local, tissue-selective inhibition of NEP should overcome the potential adverse effects of systemic, generalised NEP inhibition on other inflammatory processes.
5 Perspectives
Drug-coated stents enable site-specific delivery of beneficial compounds and much higher tissue concentrations can be achieved without the concern of systemic effects. The findings of the current study indicate that stents coated with a NEP inhibitor, or adventitial NEP inhibitor administered via a needle injection catheter, may be effective in preventing intimal hyperplasia that underlies the development of restenosis. NEP inhibitor-coated stents would increase the local concentration of the natriuretic peptides and adrenomedullin, two of the few known endogenous anti-atherogenic systems within the vascular wall.
Acknowledgements
This work was supported by the National Health and Medical Research Council of Australia through an Institute Block Grant (No. 983001) and a Dora Lush (Biomedical) Postgraduate Research Scholarship (No. 008101 for Melissa Barber). We thank Tony Dornom for the excellent care of the rabbits and Faye Docherty (Department of Anatomy and Cell Biology, University of Melbourne) for processing and staining the tissues for histology. The authors thank Dr Greg Dusting for the generous use of organ baths in his laboratory for the in vitro functional studies described in this paper.
Comments